Boosting The Anticancer Activity Of Aspergillus Favus “endophyte Of Jojoba” Taxol Via Conjugation With Gold Nanoparticles Mediated By γ‑Irradiation

Taxol Alternative Chinese Herbs Cistanche

Keywords: Aspergillus favus · Jojoba · Endophytic fungi · Gold nanoparticles · Taxol ·γ-Irradiation · Nutritional optimization

Introduction

Taxol is one of the most commercialized broad-spectrum anticancer drugs [1]. The activity of Taxol elaborates from its unique specificity for binding with the cellular tubulin β-subunits heterodimer, promoting tubulin polymerization, thus disrupting the mitotic division of tumor cells [2]. Taxol displayed a strong activity against breast, lung, head, and neck, uterine cancers, and advanced forms of Kaposi’s sarcoma [3]. Taxol was firstly produced from the bark of yew trees Taxus brevifolia “family Taxaceae” [4, 5]; however, the lower yield of Taxol that being<0.001%, i.e., to produce 1 g Taxol, it requires~10 kg of plant bark that collected from 3 to 5 trees [6], is the main challenge. In addition, the vulnerability of this plant to unpredicted fluctuations with the environmental conditions strongly influences the Taxol yield, heterogeneity, and reproducibility [7–9]. Exploring the Taxol producing potency of the endophytic fungi inhabiting medicinal plants raises the hope for overcoming the low yield by the above-mentioned method [10, 11], due to their fast growth, cost-effectiveness, independence on climatic changes, and feasibility for genetic manipulation [12, 13]. Subsequently, a plethora of endophytic fungi with metabolic potency to produce Taxol has been reported as reviewed [1, 14–25]. However, the anticipation of these fungi for industrial production of Taxol has been challenged by the attenuation and loss of Taxol productivity by the fungal storage and multiple subculturing [21, 22, 26–28]. Thus, searching for a novel fungal isolate with affordable metabolic stability and sustainability for Taxol production is the challenge. Medicinal plants of well-known ethnopharmacological relevance and traditional pharmaceutical applications could be the repertoire of novel fungal isolates with unique features of metabolic stability for Taxol biosynthesis. Among the most common medicinal plants, jojoba “Simmondsia chinensis” is a monogenetic dioecious grey-green shrub belonging to the Simmondsiaceae family. Jojoba seeds contain up to 65% of light golden and odorless high-viscosity oily metabolites [29]. Jojoba oil has been frequently used for the relief of headaches, throat inflammation, and wound treatment [30, 31]. As well as Jojoba oil has been used as an anti-inflammatory and antimicrobial agent [30, 31]. The leaves of jojoba are rich with antioxidant flavonoid compounds that are traditionally used for treating of various disorders such as asthma, inflammation, and cancer [32]. Thus, the main objective of this work was to explore a new fungal isolate from jojoba plant with unique metabolic stability for Taxol production, to evaluate the different approaches to maximize their Taxol yield, as well as, to enhance the antiproliferative activity of extracted Taxol compounds via conjugation with gold nanoparticles, mediated by gamma irradiation.

Material and Methods 

Isolation and Culturing of the Endophytic Fungi

Different parts of jojoba (Simmondsia chinensis) as leaves, barks, twigs, and buds were collected from the Faculty of Agriculture, Cairo University, and used as the source for endophytic fungi. The plant parts were collected and washed under running tap water, surface sterilized with 70% ethanol for 1 min and then rinsed with sterile water [28]. The surfacesterilized plant parts were cut into small pieces under sterile conditions and placed on plates of potato dextrose agar (PDA) medium, Czapek’s-Dox, and malt extract agar medium [33–36], and the plates were incubated at 30 °C for 10 days. The effectiveness of surface sterilization of the plant parts was assessed by centrifuging the rinsing water, then 500 μl sterile water was added to the precipitate and plated into PDA medium [37]. The purified endophytic fungal isolates were inoculated on PDA slants for 7 days and stored at 4 °C

Screening, Extraction, and Quantification of Taxol from the Endophytic Fungi 

The recovered endophytic fungi inhabiting jojoba were screened for Taxol production by growing on potato dextrose broth (PDB) [38]. One plug of each of the 7  days old fungal isolates was inoculated into 100 ml of PDB/250 ml Erlenmeyer tasks, incubated for 15 days at 30±1 °C, under shaking conditions (120 rpm). After incubation, the cultures were filtered, and the filtrate was amended with 0.2% sodium bicarbonate to precipitate fatty acids. Taxol has been extracted with dichloromethane, and the organic phase was collected and evaporated to dryness, and the residues were re-dissolved in methanol [17, 39]. Taxol was separated and identified by TLC using Merck 1 mm (20×20 cm) pre-coated silica gel plates (TLC Silica gel 60 F254, Darmstadt, Germany), detected by UV illumination at 254 nm [39]. The putative spots of Taxol were scraped-of from the TLC silica gel plates and dissolved in methanol, vortexed vigorously for 10 min, and centrifuged at 1000 rpm for 5 min. The precipitated silica particles were removed, and the supernatant was taken for Taxol quantification and purity checking by HPLC (YOUNG In, Chromass, 9110+Quaternary Pump, Korea) of C18 reverse phase column (Eclipse Plus C18 4.6×150 mm, 3.5 μm, Cat. # 959,963–902). The mobile phase used was methanol/acetonitrile/water (25:35:40, v/v/v) at a fow rate of 1.0 ml/min for 20 min [40], and Taxol fractions were measured at 227 nm, and their chemical identity and concentrations were confirmed from the retention time and absorption peak area comparing to authentic sample.

Morphological and Molecular Identification of the Recovered Endophytic Fungi

The endophytic fungal isolates were identified to their species levels based on their macro and micro-morphological features by growing on PDA, Czapek’s-Dox, and malt extract media according to the reference’s keys [33–36]. The identity of the most potent Taxol-producing fungal isolates was further molecularly confirmed based on the sequence of internal transcribed spacer (ITS) [41, 42]. Fungal genomic DNA (gDNA) was extracted by pulverizing the mycelia (~0.2 g) in liquid nitrogen, then dispensing in 1 ml CTAB extraction buffer (2% CTAB, 2% PVP40, 0.2% 2-mercaptoethanol, 20 mM EDTA, 1.4 M NaCl in 100 mM Tris−HCl, pH 8.0). The PCR primer sets were ITS4 5′-GGAAGTAAAAGTCGT AACAAGG-3′ and ITS5 5′-TCCTCCGCTTATTGATATGC-3′. The PCR reaction contains 10 μl of 2×PCR master mixture (i-Taq™, Cat. No. 25027), 2 μl of gDNA, 1 μl of each primer (10 pmol/μl), and completed to 20 μl with sterile distilled water. The PCR was programed to initial denaturation at 94 °C for 2 min, denaturation at 94 °C for 30 s, annealing at 55 °C for 10 s, extension at 72 °C for 30 s for 35 cycles, and fnal extension at 72 °C for 2 min. The PCR amplicons were analyzed by 1.5% agarose gel in 1×TBE bufer (Ambion Cat# AM9864), using 1 kb DNA ladder (Cat. # PG010-55DI) and visualized by gel documentation system. The amplicons were purified and sequenced by Applied Biosystems Sequencer, HiSQV Bases, Version 6.0 with the same primers sets. The obtained sequences were BLAST searched non-redundantly on the NCBI database, imported into MEGA 6.0 software, and aligned with the Clustal W muscle algorithm [43] and the phylogenetic tree was constructed with the neighbor-joining method of MEGA 6.0 [44].

Chemical Structure of the Extracted Taxol

The putative spots of Taxol were scraped-of from the TLC silica gel plates and purified, and the purity and concentration were determined by the UV–Vis analyses at λ 227 nm (RIGOL, Ultra-3000 Series) compared to authentic Taxol [39]. Blank media under the same conditions were used as a negative baseline for the spectrophotometric analyses. FT-IR spectrum of the purified Taxol samples was analyzed by JASCO FT-IR 3600 spectrophotometer. The Taxol sample was ground with KBr pellets, pressed into discs under vacuum, and the absorption was measured in the region 400 to 4000 cm−1 [3], compared to the authentic one. The chemical structure of extracted Taxol was confirmed from the HNMR spectroscopy (JEOL, ECA-500II, 500 MHz NMR) compared to authentic Taxol. The samples were dissolved in CDCl3, chemical shifts are given in ppm (δ-scale), and the coupling constants are expressed in hertz (Hz).

Effect of Different Types of Media on Taxol Production 

Two agar plugs (9 mm) from 7 days old cultures of each fungal isolate were inoculated in triplicate into 100  ml medium/250  ml Erlenmeyer flask of potato dextrose (PDB), Czapekʼs-Dox (CZD), M1D, and malt extract (ME) broth media. Uninoculated controls from each media that are free of fungal spores were used as a negative control, incubated at 30 °C for 15 days under the same conditions. After incubation, fungal cultures were filtered, and Taxol was extracted and determined as mentioned above.

Bioprocess Optimization of the Nutritional Conditions to Maximize the Taxol Yield

Optimization of the medium composition for maximizing the Taxol yield by the potent fungal isolate was conducted by response surface methodology using Placket-Burman design followed by central composite design [17–20, 45]. From the RSM designs, the positive and significant variables affecting Taxol production by the potent fungal isolate were assessed using the statistical software package by Design-Expert 7.0 (Stat Ease Inc., Minneapolis, USA). Each experiment was run in three biological replicates and the mean values were considered. After incubation at the desired conditions, fungal biomass was filtrated, and Taxol was extracted and quantified by TLC and HPLC as described above.

Placket‑Burman Design 

The placket-Burman design has been frequently used for the optimization of the media component for fungal growth and production of bioactive secondary metabolites, evaluating the significant variables affecting Taxol production [18, 20, 46]. The choice of factor was based on media used in qualitative and quantitative screening. Eleven factors have been included; malt extract, peptone, sucrose, soytone, glutamine, beef extract, and temperature, pH, incubation time, and shaking speed values and factors were varied over two levels, and the minimum and maximum levels ranges were selected. The statistical Design-Expert 7.0 was used to generate a set of 12 experiments. For each experiment, Taxol production was determined in three biological replicates, and the average of Taxol yield was considered. 

Regression analysis of the data was conducted using statistical software. The effect of each variable was calculated (Biometrika, 2020), using the following equation:

where, E is the effect of a testing variable, M+, and M− are the Taxol concentration of trials at that the parameter was at its higher and lower levels respectively, and N is the number of experiments that was carried out. The effect of each variable on the production was determined by calculating their respective E-values.

Where Tot high is the total responses at the high level, Tot low is the total responses at the low level, and No is the number of trials.

Central Composite Design and Interactions Between Factors Affecting Taxol Production 

The most significant positive factors affecting Taxol production by the selected fungal isolate were optimized using a response surface type CCD model experimental design [47]. By using CCD, the concentrations of the medium components were optimized, and their studied interactions were used to generate a total of 20 experiments for the three variables. To determine the optimal levels of the variables for Taxol production from the potent fungal isolate, three-dimensional (3D) response surface curves were plotted to study the interaction between the various factors and to determine the variable condition of each factor affecting Taxol production. The 3D graphs were carried out by holding three factors’ constants in an ideal level and plotting the obtained response of Taxol yield for varying levels of the other two factors.

Effect of Gamma Irradiation on Taxol Yield

The potent endophytic isolates producing Taxol were exposed to γ-irradiation with 60Cobalt source (Gamma cell 4000-A-India) at different gamma radiation doses (0.25–3.0  kGy) compared to the control of the non-irradiated culture; a dose rate 1.2 kGy/h at the time of experiments. The optimized media were inoculated by the irradiated culture under standard cultural conditions, compared to the non-irradiated spore’s inoculum as control. The cultures were incubated at 30±2 °C for 15 days on a rotary shaker (120 rpm). After incubation, the cultures were filtered and Taxol was extracted, purified, and quantified by TLC and HPLC as described above.

Synthesis and Characterization of Gold Nanoparticles (AuNPs); Conjugation with Taxol

Anticancer Activity of Taxol 

The activity of the purified Taxol and Taxol-PVP-AuNPs conjugates against liver carcinoma (HPG2), and breast carcinoma (MCF7) was determined by 3- (4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay [48]. The 96-well plate was seeded with 103 cells per well, and incubated overnight at 37 °C, then different concentrations of the drug were added, and the plates were re-incubated for 48  h. The MTT reagent (25  μl) was added, and incubated for 2  h, and the purple color of the developed formazan complex was measured at λ570 nm. The IC50 value was expressed by the amount of drug reducing the growth of 50% of an initial number of tumor cells normalizing to positive control.

Antimicrobial Activity of Taxol and Taxol‑AuNPs Conjugates

The antimicrobial activity of Taxol and Taxol-AuNPs conjugates was assessed against different bacterial isolates; Bacillus subtilis ATCC 6633 and Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, and Enterobacter agglomerans, in addition to Candida albicans. The tested bacterial cells were suspended in sterile peptone water to obtained standard inoculum of~0.5 McFarland (1–1.5)× 108  CFU/ ml at λ600 nm. The growth inhibition (mm) of microbial pathogens growth was assessed by agar disc difusion method. Sterile standard antibiotic disks with a diameter of 6.0 mm were used as positive controls. Sterile antibiotic discs (6.0  mm) were loaded with 20  μl of methanol and amoxicillin-clavulanic acid (AMC) as a negative and positive control. Discs were loaded with the same concentration of Taxol, Taxol-PVP-AuNPs, and AuNPs (1.0 μg/ml). Three biological replicates were prepared. The plates were incubated at 37 °C for 24 h, and the zones of inhibition were measured. Amoxicillin clavulanic acid (AMC) and nystatin were used to normalize the antimicrobial activity of Taxol. The inhibition zone of growth was determined by a vernier caliper (mm).

Statistical Analyses

Morphological and Molecular Identification of the Potent Taxol Producers 

Morphological features of the potent fungal isolate producing Taxol were examined according to the macroscopical and microscopical descriptive keys, as described in Materials and Methods, and revealed its morphological proximity with A. favus (Fig. 2). The fungal isolate was grown on PDA at 30 °C for 10 days and the macroscopical and microscopical features revealed its identity to such as conidial heads, mode of branching, the identity of stigma, and conidial ontology, and fruiting bodies formation, according to the universal morphological keys [33], and were found to be identical to Aspergillus favus. The potent Taxol-producing isolate A. favus was further identified based on their ITS sequences, using gDNA as a template. The PCR amplicons (~550  bp) of A. favus were resolved, purifed, and sequenced (Fig. 2). The ITS sequence of A. favus was non-redundant blast searched on NCBI database, displaying 99% similarity with A. favus, with zero E. values, and 95% query coverage. Thus, from the microscopical and molecular analyses, the target isolate was confirmed as A. favus and deposited on GenBank with accession number MW485934.1, as well as, the isolate has been deposited at Assiut University Mycological Center (AUMC), Egypt with a deposition number AUMC13892. The current isolate had 99% similarity with A. favus isolates MW485934, MT446145, KJ863514, MW522551, 

Fig. 1 A Morphological view of jojoba plant. B Plate cultures of the potent Taxol-producing endophytic fungi; A. favus Bd1 (13), A. niger Lv1 (21), Penicillium polonium (23), and A. oryzae Bd (25) on PDA after 8 days o incubation at 30 °C. The fungal isolates were grown on PDB, and incubated at the standard conditions, and Taxol was extracted and checked by TLC (C). D HPLC chromatogram of Taxol from the potent fungal isolates. E Yield of Taxol as quantified from HPLC. F, UV–Vis spectral analysis of extracted Taxol from the fungal isolates. G FT-IR analysis of extracted Taxol compared to authentic one

Fig. 2 A Macromorphological features of A. favus an endophyte of jojoba after 3, 5, and 8 days of growth on PDA. Micro-morphological features, conidial head of A. favus by 400X magnification. C PCR amplicon of A. favus ITS region of 500 bp, normalizing to 1 kb ladder (Cat.#. SM0312). D Phylogenetic analysis of ITS A. favus by maximum likelihood method [44]